Anti cd49f pe

Cervospheres were collected and placed in a 15-mL tube, where they were allowed to remain for 10 min. After some time had elapsed, supernatant was removed and the bottom sphere cells were washed with PBS and collected as previously mentioned. Pelleted cervospheres were suspended in PBS and disaggregated by mechanic pipetting. For each primary antibody, 5 × 10⁵ cells were incubated with anti-p63, anti-CK-17, Annexin II (AII) (all of these by Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in flow buffer (1X PBS, 0.05% BSA) on ice. After 30 min, cells were washed with flow buffer and spun down at 500 g (r = 11 cm) for 5 min at room temperature. Then, cells were incubated with FITC-coupled secondary antibody for 30 min on ice. After some time had elapsed, cells were once again washed in flow buffer and fixed with 4% p-formaldehyde in PBS. For CD49f detection, 5 × 10⁵ cells were incubated with anti-CD49f-PE (BD Bioscience, CA, USA) on ice for 30 min. Then, cells were washed with flow buffer and fixed. Cells were also incubated with isotype controls. Staining cells were read in BD FAC-Scan™ (BD Bioscience). At least, ten thousand events were recorded for each flow cytometer measurement. FlowJo^® software was utilized for analyzing data.

Cells were kept in culture for 72 hours. Cells collected for flow cytometry were washed with 1x PBS, trypsinised, and resuspended in PBS-2% BSA-0.05% Tween® 20. One million (1 x 10⁶) cells were used for each labelling. The primary antibodies used were: anti-CD49f-PE (BD, Pharmigen, Franklin Lakes, NJ, USA); anti-EpCAM, anti-MUC1, anti-EGFR, anti-N-cadherin, anti-E-cadherin, anti-CD66a/c/e (Biolegend, San Diego, CA, USA); anti-EpHB4 and anti-claudin-3 (R&D Systems, Inc., MN, USA); anti-vimentin, anti-claudin-4, anti-ALDH1, anti-FGFR; secondary antibodies were Alexa488 and Alexa647 (Abcam, Cambridge, UK). Unlabelled cells and with only secondary antibody labelling were included in each independent assay and considered autofluorescent. Dead cells and debris were excluded from the analysis. Cells were analyzed in a FACs-Aria flow cytometer (BD Bioscience).

Cancer stem cell markers: HeLa ML and HeLa SP cultures were suspended in flow buffer (0.05% BSA, 2 mM EDTA in PBS). For CD49f protein, the cell suspension was stained with anti-CD49f-PE (BD Biosciences, 555736) for 30 min, then washed and maintained with cold flow buffer until reading. For the ALDH assay, the Aldefluor™ kit (Stem Cell Technologies, 01700) was performed according to supplier's instructions. For the double staining with CD49f and ALDH activity, the first staining was performed with the ALDH assay followed by staining with anti-CD49f-PE which maintained these cold and protected from light until reading. The reading of the samples was done with the Beckman Coulter Cytometer (UIMEO-IMSS, México); the CD49f-positive cells were visualized by the phycoerythrin channel (FL-2), and the ALDH activity-positive cells were visualized by the fluorescein isothiocyanate channel (FL-1). The data were analyzed with FlowJo® software.